Many biological or medical phenomena are studied using optical imaging. Optical imaging, most often in the form of microscopy, can yield insights at cellular and sub-cellular levels as well as over coarser scales.
Particular proteins or other cellular components can be tagged with a fluorescent dye for study by fluorescence microscopy. Alternatively, dyes that change their fluorescence depending on calcium levels, pH, membrane voltage, or other physical or chemical characteristics can be used to report on biological phenomena such as the activity of neurons.
One frequent approach to studying these phenomena is to use wide-field illumination, often called epifluorescence. However, this optical method is not suited for studying individual cells in densely-labeled, three-dimensional tissues: these optical methods do not reject out-of-focus light, resulting in a hazy and unfocused fluorescence. The light used in these techniques is not restricted to the region of interest.
Other techniques, such as confocal or two-photon microscopy, reject out-of-focus light and achieve a higher signal-to-noise level. However, these techniques encounter serious difficulties in studying the activity of entire populations of neurons or neural circuits, largely because of their intrinsically slow rate of data collection and/or rapid photo bleaching of the sample.
An alternative to confocal and two-photon microscopy are systems that employ planar illumination. These systems often are used to image samples that are placed in a gel or other liquid substance. The samples are placed in a sample chamber, and a light source generates a light plane sideways into the sample or sample chamber, such as along a horizontal axis. A camera may be placed along a vertical axis so that it is directly above the sample chamber. The sample chamber is then rotated or otherwise moved to image sections of the sample.
This method results in several problems. First, only small samples are effectively sectioned, because the light might otherwise have to propagate through many centimeters of tissue. Also, because the optics and camera are placed above the sample and the sample must be moved to obtain sectioned images, a three-dimensional image can only be acquired slowly. Additionally, samples in the gel or other liquid move as the sample chamber moves. This can blur sample images and does not provide accurate sample images.
Consequently, new systems and methods are needed for optically studying and/or recording entire neural circuits in mammals. In addition, new systems and methods are needed for optically imaging tissues for a variety of technologies, including surgical applications and other real-time three-dimensional microscopy.